Systematic analysis of transcriptional activation from synthetic TP53 binding sites

Website guide: In the “Data Quality Checks” tab plots can be found that describe the pre-processing of the barcode counts and quality assurance. In the “Detailed Reporter Activity Analysis” tab all figures that were generated for the manuscript can be found.

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Introduction:

It is unclear how TP53 binding site architecture relates to TF activity. To test this systematically, a library was designed in collaboration with the Bussemaker lab. Design features of TP53 reporters like binding site copy number, spacer length, or core promoter choice are reviewed.

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The designed first library contains: - 6,000 TF reporters, each with up to 4 TF binding sites, followed by a minP or minCMV and a barcode in the transcription unit - 5 different TP53 motifs with different predicted binding affinities - Large range of combinatorial binding affinity - Spacer length between binding sites varied from 4-14 bp in 1 bp steps - two different core promoters - three different synthetic inactive spacer sequences - 5 barcodes per TF reporter

All TF reporters were designed using FIMO. This way, the spacings were designed to be inactive, while the TF binding sites were ensured to be intact.

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Experimental setup:

• Nucleofection into TP53-proficient MCF7 cells and TP53-KO MCF7 cells
• TP53 Stimulation with Nutlin-3a or vehicle control (DMSO)
• RNA isolation after 24h, followed by barcode-specific reverse transcription and sequencing library prep
• experiments performed in independent triplicates

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Activity quantification:

• reporter activity = cDNA counts / pDNA counts
• take average across the 5 barcodes
• then take average across the 3 biological replicates
• calculate enrichment per condition tested over background reporter activity (core promoter-only reporters)

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Repository guide:

• analysis folder: pre-processing of the barcode counts (barcode-preprocessing.Rmd) + analysis of barcode counts, linear modeling, figure generation (cDNA-processing.Rmd) + analysis of genomic TP53 response elements (mt20230623_genomic_motif_enrichment.Rmd)
• library_design folder: contains script that was used to generate the TP53 reporter sequences
• pDNA_insert_seq folder: contains scripts to analyze the full-length sequences of the plasmid pool that was used for transfections
• raw_data_analysis folder: contains the scripts that were used to extract and cluster the raw barcode counts
• TP53 binding sites were generated and scored by our collaborators from the Bussemaker lab: https://github.com/BussemakerLab/TranscriptionFactorActivityReporters